library(here)
library(tidyverse)
library(cowplot)
library(GGally)
theme_set(theme_light())

library(scuttle)
library(scater)

library(kableExtra)
library(DDCompanion)

Setup

## Directory setup
here_root <- "benchmarks/lupus"
here::i_am(file.path(here_root, "analysis/lupus-mock-results.Rmd"))
#> here() starts at /kyukon/data/gent/vo/000/gvo00063/Jeroen/DD_project_v2

res_dir <- here::here(here_root, "results")
fig_dir <- here::here(here_root, "figures")
  • Using methods: edgeR_NB, edgeR_QP, edgeR_NB_optim, edgeR_QP_optim, bGLM, qbGLM, qbGLM_offset, qbGLM_offset_squeeze
  • Using celltypes: B_mem, ncM, T4_naive
  • Using datatype: pb

Load results

res_files <- map(celltypes, ~ get_mock_res_files(
    dataset = "lupus",
    datatype = datatype,
    methods = methods,
    celltype = .x
)) %>%
    set_names(celltypes)

#res_list <- map_depth(res_files, 2, readRDS)
res_list <- lapply(res_files, function(element){
    lapply(element, readRDS)
})

## Check for expected data structure
stopifnot(unlist(map_depth(res_list, 3, ~ is.data.frame(.x$results))))

Load SCE objects

data_files <- map(celltypes, ~ get_SCE_files(
    dataset = "lupus", which = "mock_replicates",
    celltype = .x
)) %>%
    set_names(celltypes)
sce_objects <- map(data_files, readRDS)

Data overview

  • The results were generated from 5 mock replicates
  • Each replicate was generated by randomly splitting the subjects in two mock groups
  • No sub-sampling of cells per patient was performed for this data
map_dfr(sce_objects,
    ~ map_dfr(.x, function(x) c(nrows = nrow(x), ncols = ncol(x)),
        .id = "replicate"
    ),
    .id = "celltypes"
)

Subjects are divided across mock groups as follows:

map(sce_objects[[1]], function(x) table(x$ind_cov, x$mock_group))
#> $replicate_1
#>                    
#>                       A   B
#>   IGTB141_IGTB141   144   0
#>   IGTB143_IGTB143    84   0
#>   IGTB195_IGTB195     0 119
#>   IGTB469_IGTB469    89   0
#>   IGTB498_IGTB498   208   0
#>   IGTB508_IGTB508   278   0
#>   IGTB514_IGTB514   373   0
#>   IGTB645_IGTB645   123   0
#>   IGTB670_IGTB670     0  64
#>   IGTB826_IGTB826     0 511
#>   IGTB884_IGTB884   128   0
#>   IGTB986_IGTB986   137   0
#>   IGTB1372_IGTB1372   0 136
#>   IGTB1506_IGTB1506   0  83
#>   IGTB1539_IGTB1539 305   0
#>   IGTB1575_IGTB1575   0 164
#>   IGTB1650_IGTB1650   0 137
#>   IGTB1762_IGTB1762 176   0
#>   IGTB1768_IGTB1768 107   0
#>   IGTB1789_IGTB1789   0 341
#>   IGTB1793_IGTB1793   0 119
#>   IGTB1814_IGTB1814 485   0
#>   IGTB1815_IGTB1815   0 168
#>   IGTB1826_IGTB1826   0  70
#>   IGTB1827_IGTB1827 122   0
#>   IGTB1828_IGTB1828 356   0
#>   IGTB1840_IGTB1840   0 328
#>   IGTB1844_IGTB1844   0 177
#>   IGTB1871_IGTB1871   0 241
#>   IGTB1895_IGTB1895 231   0
#>   IGTB1901_IGTB1901 150   0
#>   IGTB1906_IGTB1906   0 185
#>   IGTB1908_IGTB1908 139   0
#>   IGTB1913_IGTB1913 156   0
#>   IGTB1916_IGTB1916   0 183
#>   IGTB1921_IGTB1921   0 181
#>   IGTB1952_IGTB1952 199   0
#>   IGTB1966_IGTB1966   0 567
#>   IGTB1974_IGTB1974   0 129
#>   IGTB1982_IGTB1982   0 244
#>   IGTB1987_IGTB1987   0 145
#>   IGTB1996_IGTB1996  88   0
#>   IGTB2007_IGTB2007 149   0
#>   IGTB2065_IGTB2065   0 205
#> 
#> $replicate_2
#>                    
#>                       A   B
#>   IGTB141_IGTB141   144   0
#>   IGTB143_IGTB143     0  84
#>   IGTB195_IGTB195   119   0
#>   IGTB469_IGTB469    89   0
#>   IGTB498_IGTB498     0 208
#>   IGTB508_IGTB508     0 278
#>   IGTB514_IGTB514     0 373
#>   IGTB645_IGTB645     0 123
#>   IGTB670_IGTB670     0  64
#>   IGTB826_IGTB826   511   0
#>   IGTB884_IGTB884     0 128
#>   IGTB986_IGTB986   137   0
#>   IGTB1372_IGTB1372   0 136
#>   IGTB1506_IGTB1506   0  83
#>   IGTB1539_IGTB1539 305   0
#>   IGTB1575_IGTB1575 164   0
#>   IGTB1650_IGTB1650 137   0
#>   IGTB1762_IGTB1762   0 176
#>   IGTB1768_IGTB1768 107   0
#>   IGTB1789_IGTB1789   0 341
#>   IGTB1793_IGTB1793   0 119
#>   IGTB1814_IGTB1814 485   0
#>   IGTB1815_IGTB1815 168   0
#>   IGTB1826_IGTB1826  70   0
#>   IGTB1827_IGTB1827 122   0
#>   IGTB1828_IGTB1828 356   0
#>   IGTB1840_IGTB1840   0 328
#>   IGTB1844_IGTB1844   0 177
#>   IGTB1871_IGTB1871 241   0
#>   IGTB1895_IGTB1895   0 231
#>   IGTB1901_IGTB1901 150   0
#>   IGTB1906_IGTB1906 185   0
#>   IGTB1908_IGTB1908   0 139
#>   IGTB1913_IGTB1913   0 156
#>   IGTB1916_IGTB1916   0 183
#>   IGTB1921_IGTB1921 181   0
#>   IGTB1952_IGTB1952 199   0
#>   IGTB1966_IGTB1966   0 567
#>   IGTB1974_IGTB1974   0 129
#>   IGTB1982_IGTB1982 244   0
#>   IGTB1987_IGTB1987   0 145
#>   IGTB1996_IGTB1996   0  88
#>   IGTB2007_IGTB2007 149   0
#>   IGTB2065_IGTB2065 205   0
#> 
#> $replicate_3
#>                    
#>                       A   B
#>   IGTB141_IGTB141     0 144
#>   IGTB143_IGTB143     0  84
#>   IGTB195_IGTB195     0 119
#>   IGTB469_IGTB469    89   0
#>   IGTB498_IGTB498     0 208
#>   IGTB508_IGTB508     0 278
#>   IGTB514_IGTB514     0 373
#>   IGTB645_IGTB645     0 123
#>   IGTB670_IGTB670    64   0
#>   IGTB826_IGTB826   511   0
#>   IGTB884_IGTB884   128   0
#>   IGTB986_IGTB986     0 137
#>   IGTB1372_IGTB1372 136   0
#>   IGTB1506_IGTB1506  83   0
#>   IGTB1539_IGTB1539 305   0
#>   IGTB1575_IGTB1575 164   0
#>   IGTB1650_IGTB1650   0 137
#>   IGTB1762_IGTB1762   0 176
#>   IGTB1768_IGTB1768   0 107
#>   IGTB1789_IGTB1789 341   0
#>   IGTB1793_IGTB1793 119   0
#>   IGTB1814_IGTB1814 485   0
#>   IGTB1815_IGTB1815   0 168
#>   IGTB1826_IGTB1826   0  70
#>   IGTB1827_IGTB1827 122   0
#>   IGTB1828_IGTB1828 356   0
#>   IGTB1840_IGTB1840 328   0
#>   IGTB1844_IGTB1844 177   0
#>   IGTB1871_IGTB1871   0 241
#>   IGTB1895_IGTB1895 231   0
#>   IGTB1901_IGTB1901 150   0
#>   IGTB1906_IGTB1906 185   0
#>   IGTB1908_IGTB1908 139   0
#>   IGTB1913_IGTB1913   0 156
#>   IGTB1916_IGTB1916   0 183
#>   IGTB1921_IGTB1921 181   0
#>   IGTB1952_IGTB1952   0 199
#>   IGTB1966_IGTB1966 567   0
#>   IGTB1974_IGTB1974 129   0
#>   IGTB1982_IGTB1982   0 244
#>   IGTB1987_IGTB1987   0 145
#>   IGTB1996_IGTB1996   0  88
#>   IGTB2007_IGTB2007 149   0
#>   IGTB2065_IGTB2065   0 205
#> 
#> $replicate_4
#>                    
#>                       A   B
#>   IGTB141_IGTB141     0 144
#>   IGTB143_IGTB143    84   0
#>   IGTB195_IGTB195   119   0
#>   IGTB469_IGTB469     0  89
#>   IGTB498_IGTB498     0 208
#>   IGTB508_IGTB508     0 278
#>   IGTB514_IGTB514   373   0
#>   IGTB645_IGTB645     0 123
#>   IGTB670_IGTB670    64   0
#>   IGTB826_IGTB826     0 511
#>   IGTB884_IGTB884   128   0
#>   IGTB986_IGTB986     0 137
#>   IGTB1372_IGTB1372   0 136
#>   IGTB1506_IGTB1506   0  83
#>   IGTB1539_IGTB1539 305   0
#>   IGTB1575_IGTB1575 164   0
#>   IGTB1650_IGTB1650 137   0
#>   IGTB1762_IGTB1762   0 176
#>   IGTB1768_IGTB1768 107   0
#>   IGTB1789_IGTB1789 341   0
#>   IGTB1793_IGTB1793   0 119
#>   IGTB1814_IGTB1814   0 485
#>   IGTB1815_IGTB1815 168   0
#>   IGTB1826_IGTB1826   0  70
#>   IGTB1827_IGTB1827   0 122
#>   IGTB1828_IGTB1828   0 356
#>   IGTB1840_IGTB1840   0 328
#>   IGTB1844_IGTB1844   0 177
#>   IGTB1871_IGTB1871   0 241
#>   IGTB1895_IGTB1895   0 231
#>   IGTB1901_IGTB1901 150   0
#>   IGTB1906_IGTB1906 185   0
#>   IGTB1908_IGTB1908 139   0
#>   IGTB1913_IGTB1913 156   0
#>   IGTB1916_IGTB1916 183   0
#>   IGTB1921_IGTB1921 181   0
#>   IGTB1952_IGTB1952   0 199
#>   IGTB1966_IGTB1966 567   0
#>   IGTB1974_IGTB1974   0 129
#>   IGTB1982_IGTB1982   0 244
#>   IGTB1987_IGTB1987   0 145
#>   IGTB1996_IGTB1996  88   0
#>   IGTB2007_IGTB2007 149   0
#>   IGTB2065_IGTB2065 205   0
#> 
#> $replicate_5
#>                    
#>                       A   B
#>   IGTB141_IGTB141     0 144
#>   IGTB143_IGTB143     0  84
#>   IGTB195_IGTB195     0 119
#>   IGTB469_IGTB469    89   0
#>   IGTB498_IGTB498   208   0
#>   IGTB508_IGTB508     0 278
#>   IGTB514_IGTB514   373   0
#>   IGTB645_IGTB645     0 123
#>   IGTB670_IGTB670    64   0
#>   IGTB826_IGTB826   511   0
#>   IGTB884_IGTB884     0 128
#>   IGTB986_IGTB986   137   0
#>   IGTB1372_IGTB1372 136   0
#>   IGTB1506_IGTB1506   0  83
#>   IGTB1539_IGTB1539 305   0
#>   IGTB1575_IGTB1575 164   0
#>   IGTB1650_IGTB1650 137   0
#>   IGTB1762_IGTB1762 176   0
#>   IGTB1768_IGTB1768   0 107
#>   IGTB1789_IGTB1789 341   0
#>   IGTB1793_IGTB1793   0 119
#>   IGTB1814_IGTB1814   0 485
#>   IGTB1815_IGTB1815   0 168
#>   IGTB1826_IGTB1826  70   0
#>   IGTB1827_IGTB1827   0 122
#>   IGTB1828_IGTB1828   0 356
#>   IGTB1840_IGTB1840   0 328
#>   IGTB1844_IGTB1844 177   0
#>   IGTB1871_IGTB1871   0 241
#>   IGTB1895_IGTB1895 231   0
#>   IGTB1901_IGTB1901 150   0
#>   IGTB1906_IGTB1906   0 185
#>   IGTB1908_IGTB1908   0 139
#>   IGTB1913_IGTB1913   0 156
#>   IGTB1916_IGTB1916 183   0
#>   IGTB1921_IGTB1921 181   0
#>   IGTB1952_IGTB1952   0 199
#>   IGTB1966_IGTB1966 567   0
#>   IGTB1974_IGTB1974   0 129
#>   IGTB1982_IGTB1982   0 244
#>   IGTB1987_IGTB1987 145   0
#>   IGTB1996_IGTB1996   0  88
#>   IGTB2007_IGTB2007 149   0
#>   IGTB2065_IGTB2065 205   0

Extract results of interest

Runtimes

## Get runtimes for each celltype
runtimes <- map(res_list, ~ map_dfr(.x, get_runtimes, depth = 1, .id = "method"))

P-values

res_tables <- map_depth(
    res_list, 2,
    ~ get_aggregated_rep_tables(.x, depth = 1)
) %>%
    map(combine_tables, .id = "method")

Visualize results

Run times

runtime_plots <- imap(runtimes,
    ~ plot_run_times(.x, width = 0.2, height = 0) +
      ggtitle(.y)
)

B_mem

ncM

T4_naive

Number of significant genes for different FDR levels

n_sign_summaries <- map(res_tables, function(tmp) {
    tmp %>%
        group_by(replicate, method) %>%
        summarise(
            sum(FDR < 0.01),
            sum(FDR < 0.05),
            sum(FDR < 0.1),
            .groups = "drop"
        )
})

B_mem

Cells per patient: B_mem
method sum(FDR < 0.01) sum(FDR < 0.05) sum(FDR < 0.1)
Replicate: 1
bGLM 9 13 16
edgeR_NB 0 0 0
edgeR_NB_optim 0 0 0
edgeR_QP 0 0 0
edgeR_QP_optim 0 0 0
qbGLM 0 0 0
qbGLM_offset 0 0 0
qbGLM_offset_squeeze 0 0 0
Replicate: 2
bGLM 25 66 104
edgeR_NB 0 0 0
edgeR_NB_optim 0 0 0
edgeR_QP 0 0 0
edgeR_QP_optim 0 0 0
qbGLM 0 1 1
qbGLM_offset 0 0 1
qbGLM_offset_squeeze 0 0 0
Replicate: 3
bGLM 10 23 56
edgeR_NB 0 1 1
edgeR_NB_optim 0 1 1
edgeR_QP 0 1 1
edgeR_QP_optim 0 1 1
qbGLM 0 0 0
qbGLM_offset 0 0 0
qbGLM_offset_squeeze 0 0 0
Replicate: 4
bGLM 12 20 27
edgeR_NB 0 0 0
edgeR_NB_optim 0 0 0
edgeR_QP 0 0 0
edgeR_QP_optim 0 0 0
qbGLM 0 0 0
qbGLM_offset 0 0 0
qbGLM_offset_squeeze 0 0 0
Replicate: 5
bGLM 7 19 26
edgeR_NB 0 0 0
edgeR_NB_optim 0 0 0
edgeR_QP 0 0 0
edgeR_QP_optim 0 0 0
qbGLM 0 0 0
qbGLM_offset 0 0 0
qbGLM_offset_squeeze 0 0 0

ncM

Cells per patient: ncM
method sum(FDR < 0.01) sum(FDR < 0.05) sum(FDR < 0.1)
Replicate: 1
bGLM 4 29 38
edgeR_NB 0 0 0
edgeR_NB_optim 0 0 0
edgeR_QP 0 0 0
edgeR_QP_optim 0 0 0
qbGLM 0 0 0
qbGLM_offset 0 0 0
qbGLM_offset_squeeze 0 0 0
Replicate: 2
bGLM 17 33 53
edgeR_NB 0 0 0
edgeR_NB_optim 0 0 0
edgeR_QP 0 0 0
edgeR_QP_optim 0 0 0
qbGLM 0 0 0
qbGLM_offset 0 0 0
qbGLM_offset_squeeze 0 0 0
Replicate: 3
bGLM 19 28 43
edgeR_NB 0 1 1
edgeR_NB_optim 0 1 1
edgeR_QP 0 1 1
edgeR_QP_optim 0 1 1
qbGLM 0 0 0
qbGLM_offset 0 0 0
qbGLM_offset_squeeze 0 0 0
Replicate: 4
bGLM 68 154 296
edgeR_NB 1 1 2
edgeR_NB_optim 0 1 3
edgeR_QP 1 2 2
edgeR_QP_optim 0 1 3
qbGLM 1 1 1
qbGLM_offset 1 1 1
qbGLM_offset_squeeze 1 1 1
Replicate: 5
bGLM 6 15 26
edgeR_NB 0 0 0
edgeR_NB_optim 0 0 0
edgeR_QP 0 0 0
edgeR_QP_optim 0 0 0
qbGLM 0 0 0
qbGLM_offset 0 0 0
qbGLM_offset_squeeze 0 0 0

T4_naive

Cells per patient: T4_naive
method sum(FDR < 0.01) sum(FDR < 0.05) sum(FDR < 0.1)
Replicate: 1
bGLM 64 125 177
edgeR_NB 0 0 0
edgeR_NB_optim 0 0 0
edgeR_QP 0 0 0
edgeR_QP_optim 0 0 0
qbGLM 0 0 0
qbGLM_offset 0 0 0
qbGLM_offset_squeeze 0 0 0
Replicate: 2
bGLM 75 167 277
edgeR_NB 0 0 0
edgeR_NB_optim 0 0 0
edgeR_QP 0 0 0
edgeR_QP_optim 0 0 0
qbGLM 0 0 0
qbGLM_offset 0 0 0
qbGLM_offset_squeeze 0 0 0
Replicate: 3
bGLM 64 152 220
edgeR_NB 0 0 0
edgeR_NB_optim 0 0 0
edgeR_QP 0 0 0
edgeR_QP_optim 0 0 0
qbGLM 0 0 0
qbGLM_offset 0 0 0
qbGLM_offset_squeeze 0 0 0
Replicate: 4
bGLM 78 149 233
edgeR_NB 0 0 0
edgeR_NB_optim 0 0 0
edgeR_QP 0 0 0
edgeR_QP_optim 0 0 0
qbGLM 0 0 0
qbGLM_offset 0 0 0
qbGLM_offset_squeeze 0 0 0
Replicate: 5
bGLM 65 117 172
edgeR_NB 0 0 0
edgeR_NB_optim 0 0 0
edgeR_QP 0 0 0
edgeR_QP_optim 0 0 0
qbGLM 0 0 0
qbGLM_offset 0 0 0
qbGLM_offset_squeeze 0 0 0

P-value distributions

pval_figs <- map(res_tables, pval_hist)

B_mem

ncM

T4_naive

P-value distributions stratified by expression level

For each replicate, calculate average log-normalized counts, using scuttle::calculateAverage().

## Calculate average counts for each replicate
avg_counts <- map_depth(sce_objects, 2,
    ~ calculateAverage(counts(.x))
)

## Split res_tables per replicate
res_tables_split <- map(res_tables, ~ split(.x, .x$replicate))

## Add average counts to results tables
## Note that for the 'combined' celltypes, the average counts will be the same
## for each celltype contrast
res_tables_split <- map2(
    res_tables_split, avg_counts,
    ~ map2(.x, .y, function(res, avg_cnt) {
        res$avg_count <- avg_cnt[res$gene]
        res
    })
)

Make p-value histograms stratified by the average counts.

pval_hist_strat_plots <- map(res_tables_split,
    ~ imap(.x, function(y, replicate) {
        replicate <- str_remove(replicate, "replicate_")
        title <- paste("Replicate:", replicate)
        pval_hist_strat(y, n_groups = 6) +
            ggtitle(title)
    })
)

B_mem

ncM

T4_naive

Session info

Session info
#> [1] "2023-05-27 16:04:24 CEST"
#> ─ Session info ───────────────────────────────────────────────────────────────
#>  setting  value
#>  version  R version 4.2.0 (2022-04-22)
#>  os       Red Hat Enterprise Linux 8.6 (Ootpa)
#>  system   x86_64, linux-gnu
#>  ui       X11
#>  language (EN)
#>  collate  en_US.UTF-8
#>  ctype    en_US.UTF-8
#>  tz       Europe/Paris
#>  date     2023-05-27
#>  pandoc   2.13 @ /apps/gent/RHEL8/zen2-ib/software/Pandoc/2.13/bin/ (via rmarkdown)
#> 
#> ─ Packages ───────────────────────────────────────────────────────────────────
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#>  P beachmat               2.14.2    2023-04-07 [?] Bioconductor
#>  P beeswarm               0.4.0     2021-06-01 [?] CRAN (R 4.2.0)
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#>  P BiocManager            1.30.20   2023-02-24 [?] CRAN (R 4.2.0)
#>  P BiocNeighbors          1.16.0    2022-11-01 [?] Bioconductor
#>  P BiocParallel           1.32.6    2023-03-17 [?] Bioconductor
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#>  P cli                    3.6.1     2023-03-23 [?] CRAN (R 4.2.0)
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#>  P DT                     0.28      2023-05-18 [?] CRAN (R 4.2.0)
#>  P edgeR                  3.40.2    2023-01-19 [?] Bioconductor
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#>  P ── Loaded and on-disk path mismatch.
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